The relaxation of airway smooth muscle is governed in part by agonist binding to G-protein coupled Beta-adrenergic receptors, principally of the Beta2-subtype (Beta2AR). Agonist-activated Beta2ARs desensitize within minutes by rapid phosphorylation and endocytosis, then apparently resensitize by dephosphorylation and recycling back to the cell surface. Prolonged agonist exposure of many hours causes a severe attenuation of responsiveness to Beta-agonist, a large part of which is due to a loss of cellular Beta2ARs, termed downregulation. It is important to understand this process because of the widespread use of Beta2-agonist drugs in therapy for asthma and chronic obstructive pulmonary disease, and the possibility that the efficacy of these drugs may be reduced due to the downregulation of Beta2ARs. Our goal in this proposal is to determine how receptor phosphorylation and endocytosis is related to receptor downregulation. It seems likely that distinct structural features of the Beta2AR are involved in determining the extent of downregulation, since this function is affected by some receptor mutations. However, it is unclear what if any role endocytosis plays in receptor downregulation. To approach this question, we will systematically vary the rate of receptor endocytosis by blocking the formation of endocytic coated vesicles with dynamin dominant negative expression. The endosome concentration of receptors will be varied by constructing cell lines expressing different levels of receptors. The effects of these manipulations on receptor degradation and downregulation will be quantitatively examined to test several models of receptor downregulation. Also, because endocytosis of receptor may be required for its resensitization, we will examine receptor phosphorylation under conditions where endocytosis is inhibited. Finally, we will examine the endocytic pathways used by mutant receptors that downregulate but do not apparently undergo rapid internalization, and receptors that do not downregulate despite their rapid internalization. Completion of these aims will move us significantly closer towards our long-term goal of determining the mechanism of Beta2AR downregulation.